KR20150055016A - Detection of non-nucleic acid analytes using strand displacement exchange reactions - Google Patents

Abstract

The present invention relates to a detection system for DNA and RNA and other analyte detection. The system includes a set of oligonucleotides that can hybridize to each other in a specific way and generate signals based on specific hybridization phenomena. The system relies on changes in the hybridization equilibrium between the oligonucleotides in the presence of the analyte or analyte causing the signal change.

Description

DETECTION OF NON-NUCLEIC ACID ANALYTICS USING STRAND DISPLACEMENT EXCHANGE REACTIONS < RTI ID = 0.0 >

The present invention relates to an analyte detection system. In particular, the invention relates to an analyte detection system, wherein the analyte is an analyte that is not a nucleic acid, i.e., DNA and RNA.

 Various other methods, such as ELISA, SPR, QCM, electrochemical sensors, etc., can be used to detect peptides / proteins and other molecules in the sample. While these surface-based methods are given high sensitivity and multiplex sensing ability, the immobilization process can interfere with protein-ligand binding and frequently requires blocking to prevent careful washing and nonspecific adsorption. There are few well-established homogenization methods, and fluorescence polarization is commonly used for the study of small molecule-protein interactions. (Analysis of protein-ligand interactions by fluorescence polarization, Ana Rossi & Colin Taylor, Nature protocols, 2011).

Electrochemical biosensors are generally based on enzyme-catalysed reactions that produce or consume electrons. Enzyme-linked immunosorbent assays (ELISAs) are plate-based assays designed to detect and quantify substances such as peptides, proteins, antibodies and hormones. Surface plasmon resonance (SPR) detects the increase in fixed ligand size by recording changes in the refractive index of the surface of a thin metal film when it binds a larger protein. The quartz crystal microbalance (QCM) detection method is based on measurement of mass change and physical properties of thin films deposited on crystal surfaces. Fluorescence polarization detects the binding of small fluorescent ligands to larger proteins using plan-polarized light to detect changes in effective molecular volume.

Various assays such as PCR, Southern blotting, Northern blotting and FISH allow detection of nucleic acid molecules in the sample. Moreover, a recent approach is the DNA tohold exchange reaction. A DNA toehold exchange reaction is a system in which two DNA strands each having a specific tohod region compete with each other to hybridize to a third DNA strand serving as a substrate. Due to its high modularity, designability and sensitivity, it has developed a catalytic DNA network (Zhang et al., Engineering entropy-driven reactions and networks catalyzed by DNA. Science 2007, 318, 1121-1125) Strand displacement kinetic (Zhang et al., Control of DNA strand displacement kinetics using toehold exchange, J Am Chem Soc 2009, 131, 17303-17314), optimization of nucleic acid hybridization specificity (Zhang et al., Optimization of specificity of nucleic acid Nat Chem 2012, 4, 208-214). However, such a system is only suitable for detection of nucleic acid molecules.

Thus, improved detection systems for detecting DNA and other molecules as well as RNA are advantageous, and more efficient and / or reliable detection systems for detecting DNA and RNA and other small molecules will be advantageous.

The present invention provides an analyte detection system for detecting DNA and RNA and other analytes. The system includes a set of oligonucleotides that can hybridize to each other in a specific manner and can generate signals based on specific hybridization phenomena. The system relies on hybridization equilibrium changes between oligonucleotides in the presence of the analyte or analyte causing the signal change.

Provided herein is a detection system that utilizes a DNA tohold exchange reaction for non-nucleic acid target detection by attaching small molecules (e. G., Antigen or hapten) to one or more of the three DNA strands. The specific binding between the small molecule and its receptor protein or antibody shifts the original equilibrium and the population change of each component in the solvent can be monitored, for example, by FRET (Forster resonance energy transfer). The scheme is shown below:

Where A, B, and S are three DNA oligonucleotides, where A and b are partially complementary to S. X represents a small molecule labeled with A, and Y is a corresponding binding protein. For example, in the absence of Y, A and B have the same or nearly the same ability to hybridize to S, and if Y is present, the bulkiness, charge or other effects of the protein will affect the hybridization energy Given that the ratio can vary from 50:50 to 20:80, for example. Basically, this concept exploits the fact that for each hybrid formation a small perturbation of energy causes a relatively large shift in the equilibrium between the two DNA hybrids.

In general, the tohod exchange reaction system shares two identical point-of-motion regions, but the two DNA strands that differ in the tohod region compete with each other dynamically to hybridize with the third DNA. The total substitution cycle is shown in Fig. For example, strand A (A) is paired with strand S (S) to initially form an AS duplex. Since S has a different to-hold region with respect to strand B (B), the tohole of B has an opportunity to hybridize with the tohole of S. As soon as both A and B bind to S, due to sequence symmetry, a branch point transfer process occurs. In this process, A and B have the same opportunity to substitute each other, so that A only bonds with S through the tohtow, while half of the product can be that B has a complete branching point region of S . Because the tohole is generally short, hybridization is unstable and transient, resulting in the final leaving of the components of BS and A in solution. All of the above steps are reversible. Note that B generally has a binding moiety labeled in the point-of-travel region.

In the presence of the analyte, the binding of the analyte to the small molecule bound to B may not have a significant effect on the tohound binding, but may cause an increased energy barrier for B performing the branching point transfer process, Thus giving B a disadvantage (or sometimes an advantage) as compared to A, which competes with S. Thus, in the final product, the thermodynamic equilibrium can be shifted in the direction of forming more AS duplexes (often more BS duplexes) as well as single-strand B bound to the target analyte, Can be detected by FRET or other optical methods.

In another type of study, the Example portion shows that the concept actually works for RNA and DNA and other analyte detection.

 In conclusion, a novel detection system has been developed that can detect both proteins and small molecules on the basis of a fine-tuned tohod exchange reaction. Compared to conventional methods such as ELISA, this method is enzyme-free and avoids protein modification and bivalent antigen / antibody screening efforts.

Such a method can find use as a sensitive, specific, and robust high-throughput platform to detect activity associated with health, food, animals and the environment.

Accordingly, an object of the present invention is to provide a detection system capable of detecting DNA and RNA and other molecules. Another object is to provide a detection system capable of detecting DNA and RNA and other small molecules and requiring only one binding site in the analyte to detect the analyte. This is in contrast to ELISA assays, which require, for example, two binding sites in the analyte. Two binding sites may not be present in the small molecule.

Accordingly, one aspect of the present invention relates to an analyte detection system for detecting an analyte other than DNA and RNA, the system comprising a minimal first oligonucleotide A, a first oligonucleotide B and a third oligonucleotide S , Where:

Each of oligonucleotide A and oligonucleotide B comprises a complementary or partially complementary sequence to the sequence of oligonucleotide S wherein oligonucleotides A and B compete for hybridization with oligonucleotide S in dynamic equilibrium, Optionally, at least one of the oligonucleotides A and B comprises a covalently linked binding moiety capable of interacting with DNA and RNA and other analytes;

The covalently linked binding moiety, which is bound to one or more of the oligonucleotides A and B, or to the oligonucleotide, is capable of interacting with DNA and RNA and other analytes to form oligonucleotides or binding moieties The interaction of the te and the analyte causes a shift of the hybridization equilibrium, which provides a detectable signal.

An illustrative embodiment of the analyte detection system is shown in FIGS. 1 and 2. FIG. As described in the background, the assay is similar to the assay described for DNA detection. However, the setup is not suitable for detecting DNA and RNA and other analytes, such as proteins and small organic molecules. Different systems such as ELISA are more suitable for this purpose.

Another aspect of the invention relates to a kit of parts comprising an analyte detection system in accordance with the present invention.

Another aspect of the invention provides a kit of parts comprising a first oligonucleotide, a second oligonucleotide and a third oligotreotide in accordance with the present invention.

Another aspect of the present invention provides a method for detecting the presence or level of an analyte and other DNA and RNA in a sample,

comprising the steps of: a) providing a sample containing or suspected of containing an analyte of interest;

b) providing an analyte detection system in accordance with the present invention;

c) incubating the sample in the analyte detection system;

d) comparing the reference level with the detected level of the analyte; And

e) measuring the presence or level of the analyte in the sample.

1
Figure 1 illustrates a particular embodiment of the invention for a particular sequence of a first oligonucleotide 1 (SEQ ID NO: 1), a second nucleotide 3 (SEQ ID NO: 2) and a third nucleotide 5 (SEQ ID NO: 3) For example. 8, 8 'and 9, 9' represent toehold regions, and 7, 7 'represent branch migration regions. The arrows indicate the direction from the 5 'to 3' of the oligonucleotide.
2
Figure 2 shows an example of how the equilibrium changes when the binding moiety 4 and the analyte are combined. The numbers are as shown in Fig. a) When there is no target analyte, A and B are designed to have the same or near-same probability to hybridize with S, resulting in a 50/50 ratio for AS and BS. b) The stereoscopic or electrostatic effect of the non-analyte (in this case the target) in the presence of the analyte results in an energetic barrier to B performing the branch point transfer process, Causing an 80/20 ratio.
3
Figure 3 shows a detection system when biotin is a binding moiety and streptavidin (STV) is the analyte. (SEQ ID NO: 4), a second oligonucleotide 3 (SEQ ID NO: 5) and a third oligonucleotide 5 (SEQ ID NO: 6) according to the present invention. The numbers are as shown in Fig. Also, 2 and 6 represent the signal system and 4 represents the coupling moiety.
4
Figure 4 shows the results of a three-stranded system for streptavidin (STV) detection and biotin detection. (a) Normalized FRET effect for each of two samples in the presence or absence of STV. (b) Fluorescence spectrum for each of the two samples in the presence or absence of STV. (c) The titration curve of STV detection. (d) Non-denaturing gel analysis for STV detection method. The front two lanes are systems containing one biotin and the remaining two lanes are two biotin-containing systems. An inset is a scheme of STV bound on the B strand by a two-site correlation. (e) quantitative detection of biotin by a low-risk strategy. First, the analyte (biotin) is mixed with STV and the mixture is then included in the assay. The analyte occupies the binding site in the STV and interacts accordingly.
5
Figure 5 Three strategies for sensor analysis are presented. In Strategy 1, the direct detection of the analyte / target (e.g., protein or antibody) is obtained by interaction with the binding moiety in the ABS system. In Predatory Strategy 2, unknown samples and corresponding proteins are premixed and subsequently added to a general assay. If the sample contains a free ligand, it limits the binding site of the protein, rendering it unable to function as an assay. In Competitive Strategy 3, the protein is first mixed with conventional DNA assays for the formation of a new assay, then added to compete with the ligand of strand B to bind an unknown sample to the protein, and thus has the opposite effect on equilibrium .
6
Figure 6 shows a control experiment. A) Influence of additive in the ABS system described in FIG. 3 without bond moiety. Three fluorophore setups. B) The effect of the additive in the ABS system described in FIG. 3, with a biotin bond moiety. Two fluorophore setups. No detectable change is observed after addition of any analyte except in the case of adding streptavidin to a system containing a biotin binding moiety.
7
Figure 7 shows the kinetic of streptavidin binding assay. Compare the two analyzes for each of the 4 nt (left) and 6 nt (right) lengths of the torpedo, respectively. In the quantitative analysis, the biotin strain is located in the tohred region of B, and A and S have a pair of fluorophore (Alexa488 & Alexa555). This result indicates that in the expense leading to the less effect of STV coupling, the longer to hold (in both A and B) guarantees a faster equilibrium, as can be seen in the results of FRET in (b). The final design preferably compromises the kinetic and signal-to-noise ratios. In addition, the FRET analysis shows that the 3 hour incubation is sufficient for a 4 nt system reaching equilibrium after STV addition (data not shown).
8
Figure 8 shows a detection system when digoxigenin (DIG) is a binding moiety and anti-digoxigenin (aD) is the analyte. Figure A) shows a specific sequence comprising a modification of the first oligonucleotide 1 (SEQ ID NO: 7), the second oligonucleotide 3 (SEQ ID NO: 8) and the third oligonucleotide 5 (SEQ ID NO: 6) . The numbers are as shown in Fig. Also, 2 and 6 represent the signal system and 4 represents the coupling moiety. Bar figure B) shows the raw FRET signal change during aD detection. C) The chemical structure of dioxygen. Graph D) shows FRET signal changes upon titration of aD against oligonucleotides 1, 3 and 5. E) shows the intrinsic fluorescence image of polyacrylamide gel in which only oligonucleotide A is visible. The increase in the AS group is seen with addition of aD.
9
Figure 9 shows a detection system of human plasma when digoxigenin (DIG) is the binding moiety and anti-digoxigenin (aD) is the analyte. Figure A) Numbers are the same as Figure 8A. Graph B) is the result of three controlled experiments comparing the sensor (ABS) setup of a sample with no added human plasma or added human plasma. Columns 1 and 3 show RET changes upon detection of human plasma-free aD, and 2 and 5 show FRET changes in human plasma. Columns 4 and 6 show competitive assays for the detection of each free digoxigenin with or without human plasma.
10
Figure 10 shows the detection system of human saliva when digoxigenin (DIG) is the binding moiety and anti-digoxigenin (aD) is the analyte. Figure A) Numbers are the same as Figure 8A. Bar Fig. B) shows the change of the AS group when detecting aD in the saliva sample.
11
Figure 11 shows a detection system when the various digoxigenin (DIG) molecules act as binding moieties and anti-digoxigenin (aD) is the analyte. Figure A) shows a specific sequence comprising a modification of a first oligonucleotide 1 (SEQ ID NO: 7), a second oligonucleotide 3 (SEQ ID NO: 8) and a third oligonucleotide 5 (SEQ ID NO: 6) . Figure 11 also shows various DIG moieties for the coupling of aD. Figure B) shows changes in the AS group with detection of aD.
12
Figure 12 shows the detection system when operating as a competitive / degraded sensor. Here, digoxigenin (DIG) along with anti-digoxigenin (aD) acts as the binding moiety and free DIG acts as the analyte. Figure A) is the same as Figure 8A. Bar Figure B) shows the AS population change in the hypoxic assay for detecting free DIG in buffers, human plasma and human saliva.
13
FIG. 13 shows the DIG titration curve by a low-resolution assay or a competitive assay.
14
Figure 14 shows the detection system when vitamin D (vitamin D (VD)) is the binding moiety and vitamin D-binding protein (DBP) is the analyte. Moreover, it represents free vitamin D as a target in low-cost and competitive assays. The model shows the substitution of strands of B that are helped by the binding of DBP to the VD of B. The band transfer assay shows a fluorescence image of a native polyacrylamide gel in which only oligonucleotide A is visualized. The increase of the AS group is seen as the addition of DBP. The first graph shows the DBP titration for the sensor. The second and third graphs represent the degradation and competitive detection of VD, respectively.
15
Figure 15 shows the design and results of a three-stranded aptamer system for DNA detection. Refers to a specific sequence comprising a modification of a first oligonucleotide 1 (SEQ ID NO: 10), a second oligonucleotide 3 (SEQ ID NO: 11) and a third oligonucleotide 5 (SEQ ID NO: 12) according to the present invention. The numbers are as shown in Figs. (A) Schematic of ATP detection by structure-switching aptamer. Binding of ATP can reduce the burden of B to hold, thereby hindering BS formation. (B) ATP detection titration curve.
16
Figure 16 shows the system when a truncated DNA peroxidase signaling system is used. (SEQ ID NO: 13), a second oligonucleotide 3 (one biotin: SEQ ID NO: 5 and two biotin SEQ ID NO: 14) and a third oligonucleotide 5 (SEQ ID NO: 15) according to the present invention, ). ≪ / RTI > The numbers are as shown in Figs. The two-biotin system is even capable of visual inspection. And detection of one biotin and two biotin. (A) schematic and results of STV detection by a truncated DNA peroxidase signaling system. When two biotins are bound to B, the color change in the presence of STV is evident even to the naked eye. (B) the absorbance of one or two biotin samples in the presence or absence of STV.
17
Figure 17 shows a one-strand system design according to the present invention. The numbers are as shown in Figs. 11 denotes a linker area.
18
Figure 18 shows the one-stranded system for STV detection and the results obtained using the sequence shown in Example 12. (a) Schematic of STV detection using a one-stranded system. (B) the absorbance of a single-stranded sample in the presence or absence of STV. ≪ tb >< TABLE > Id = Table 2 Columns = 3 < tb > .
19
Figure 19 shows a strategy for the detection of a particular small molecule or ion. (a) Melanin detection scheme by making use of bifacial melamine-thymine recognition through hydrogen-binding (insertion). In the absence of melanin, strand A has a longer to hold than B, so it has a higher priority to combine with S. (b) (a) using the same set-up as shown and specificity and the TT mismatch site (mismatch sites) of the DNA enemy "sandwich" so as to form a complex Hg ^{2 +} is well known, mercury divalent cation (mercury bivalent cation) can be detected, and Hg ^{2 +} is linearly bonded to Nl nitrogen of the thymine residue on each side (insert).
20
Figure 20 shows a more advanced strategy when the strand S has one (a) or two (b) hairpins in it. The function of the inner hairpin is to create a larger steric hindrance by a three-dimensional configuration to prevent hybridization between S and B of the bound protein, And a multi-arm junction around the location of its binding protein.
The present invention can be described in more detail below.

Analyte  Detection system ( Analyte detection system )

One aspect of the invention is based on the hybridization equilibrium between the three oligonucleotides and relates to an analyte system capable of detecting non-nucleic acid analytes of a sample.

One embodiment of one aspect of the present invention is an analyte detection system comprising a first oligonucleotide (1), a second oligonucleotide (3), and a third oligonucleotide (5)

The first or second oligonucleotide comprises a first group (2) forming a first part of the signal system;

The third nucleotide comprises a second group (6) forming a second part of the signal system;

At least one covalently bonded moiety (4) is located on the first or second oligonucleotide;

Wherein hybridization between the first or second oligonucleotide and the third oligonucleotide may produce a signal or may facilitate the generation of a different signal than when the first or second oligonucleotide and the third oligonucleotide are not hybridized;

Wherein the presence of the analyte changes the hybridization equilibrium of the detection system and causes a change in signal.

The analyte detection system can be, for example, one here:

The first oligonucleotide (1)

A first tohold region 8 located at the 5'-plane of the branch migration region 7;

The second oligonucleotide (3)

A second toothed region (9) located on the 3'-plane of the minute shift region (7);

The third oligonucleotide (5)

A first tohold region 8 ';

A second toe hold region 9 '; And

And a minute shift region 7 '.

here:

The first to fourth region 8 of the first oligonucleotide 1 and the first toe region 8 ' of the third oligonucleotide 5 comprise a complementary sequence;

The splitting point shifting region 7 of the first oligonucleotide 1 and the splitting point shifting region 7 'of the third oligonucleotide 5 comprise a stretch of complementary nucleotides;

The second toothed region 9 of the second oligonucleotide 3 and the second toothed region 9 'of the third oligonucleotide 5 comprise an extension of complementary nucleotides;

The branching point shifting region 7 of the second oligonucleotide 3 and the branching point shifting region 7 'of the third oligonucleotide 5 comprise an extension of the complementary nucleotide.

In certain embodiments, the invention is an analyte detection system for detecting DNA and RNA and other analytes,

- the first oligonucleotide (1) is

A first toothed region 8 located on the 5'-plane of the branch migration region 7;

Optionally one or more covalently bonded moieties (4);

A first group (2) that optionally forms a first part of the signaling system;

- the second oligonucleotide (3) is

A second tohold region 9 located on the 3'-plane of the branching point moving region 7;

Optionally one or more covalently bonded moieties (4);

A first group (2), optionally forming a first part of the signal system,

- the third oligonucleotide (5)

A first tohold region 8 ';

A second tohold region 9 ';

A minute point moving region 7 ';

Optionally one or more covalently bonded moieties (4);

A second group (6) forming a second part of the signaling system;

The first group (2), which conditionally forms the first part of the signal system, comprises a first oligonucleotide (1) and / or a second oligonucleotide (3);

Conditionally, the at least one covalently bonded moiety (4) is located in the first oligonucleotide (1) and / or the second oligonucleotide (3) and / or the third oligonucleotide (5);

Wherein the first to fourth region (8) of the first oligonucleotide (1) and the first to fourth region (8 ') of the third oligonucleotide (5) comprises a complementary sequence;

Wherein the junction point shifting region 7 of the first oligonucleotide 1 and the junction point shifting region 7 'of the third oligonucleotide 5 comprise a stretch of complementary nucleotides;

Wherein the splitting point shifting region (7) of the second oligonucleotide (3) and the splitting point shifting region (7 ') of the third oligonucleotide (5) comprise an extension of the complementary nucleotide;

Wherein the second toe region (9) of the second oligonucleotide (3) and the second toe region (9 ') of the third oligonucleotide (5) comprise an extension of the complementary nucleotide;

Wherein the hybridization between the first oligonucleotide (1) and the third oligonucleotide (5) produces a signal or a signal that is different from the case where the first oligonucleotide (1) and the third oligonucleotide (5) Wherein the first group (2), which conditionally forms the first part of the signal system, comprises a first oligonucleotide (1);

Wherein the hybridization between the second oligonucleotide (3) and the third oligonucleotide (5) produces a signal, or a signal generation different from the case where the second oligonucleotide (3) and the third oligonucleotide (5) , And wherein the first group (2), which conditionally forms the first part of the signal system, comprises a second oligonucleotide (3).

As used herein, the term "detection" includes quantitative determination of the amount of analyte using the present invention, as well as qualitative detection in the presence or absence of the analyte.

Reference to "analyte ", as used herein, includes two or more analytes applicable, as well as the detection of a single analyte.

The oligonucleotides A, B and S may usually be isolated nucleotide strands, but the invention may also be carried out with two or more oligonucleotides connected in part or in whole by covalent bonds.

Further, the present invention is generally described herein using three oligonucleotides, generally referred to as A, B, or S, for simplicity, whereas the method is characterized in that additional oligonucleotides C or two additional oligonucleotides C and D , And additional oligonucleotides, as described herein, may be similar to oligonucleotides A and / or B. The term " oligonucleotide A "

Alternatively or additionally, it can be performed with one or more oligonucleotides similar to oligonucleotides S as described herein. According to an embodiment, the present invention can be carried out together with a first set oligonucleotide (A, B, S) for the detection of a first analyte and a second set oligonucleotide for detection of a second analyte (C , D, S ').

Another alternative is that the oligonucleotide S comprises at least one hybridization domain. An example of an alternative is shown in Figure 20, where S represents having one or more hairpin turns, thereby forming multiple hybridization domains. A multiple hybridization domain that is part of a single oligonucleotide S is shown in Figure 20, and it is also possible to use a configuration in which two or more such binding domains are located on separate nucleotide strands.

As mentioned earlier, the system is based on a change in the hybridization equilibrium between the oligonucleotides in the system. Thus, in embodiments of the invention, the presence of the analyte changes the hybridization equilibrium of the detection system resulting in a signal change compared to when the analyte is not present. Thus, the present invention is a unique system that utilizes a hybridization phenomenon that occurs between oligonucleotides (e. G., DNA) that detects the presence or level of non-DNA (or non-RNA) This assay has several advantages compared to, for example, ELISA assays.

- It requires very little "hands-on" and is a fast and inexpensive method. The component is stable for a long time.

- The method can be performed under isothermal conditions, and inexpensive equipment is required.

- Small analytes can be analyzed more easily because only one binding to the analyte occurs. This can be compared, for example, to an ELISA in which two binding sites are required in the analyte.

- the method is homogeneous and thus avoids immobilization and washing processes as well as non-specific adsorption problems.

- The assay can avoid antibody markers or protein modifications.

The first oligonucleotide ( First 자이클립 )

The first oligonucleotide (1) forms part of the detection system. In one embodiment, the length of the first oligonucleotide (1) is 10-100, 15-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, Such as 90-100, 8-90, 8-80, 8-70, 8-60, 8-50, 8-40, 8-30, 8-20 or 8-15. In another embodiment, the first oligonucleotide (1) is selected from SEQ ID NOS: 1, 4, 7, 10 and 13. Even if a specific sequence is provided, other sequence combinations can be selected, so that the present invention is not limited to the above sequence. It is emphasized that the analyte is detected independently of the sequence. Other sequences may be useful if multiple assays are designed for the detection of one or more samples of the analyte. The example portion shows the result of another set of oligonucleotides.

The term "5'-face" in this context means a nucleic acid sequence located at a specific point or a 5 'portion of a region containing a nucleic acid molecule. Similarly, the term "3'-face" refers to a nucleic acid sequence located at the 3 ' portion of a particular point or region comprising a nucleic acid molecule.

The second oligonucleotide ( Second  oligonucleotide)

The second oligonucleotide (3) forms part of the detection system. In one embodiment, the length of the second oligonucleotide (3) is 10-100, 15-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, Such as 90-100, 8-90, 8-80, 8-70, 8-60, 8-50, 8-40, 8-30, 8-20 or 8-15 nucleotides. In another embodiment, the second oligonucleotide (3) is selected from the group consisting of SEQ ID NOS: 2, 5, 8, 9, 11 and 14. As mentioned above, the present invention is not limited to this sequence.

Third  Oligonucleotides ( Third  oligonucleotide)

The third oligonucleotide (5) forms part of the detection system. In one embodiment, the length of the third oligonucleotide (5) is 10-100, 15-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, Such as 90-100, 8-90, 8-80, 8-70, 8-60, 8-50, 8-40, 8-30, 8-20 or 8-15 nucleotides. In yet another embodiment, the third oligonucleotide (3) is selected from the group consisting of SEQ ID NOS: 3, 6, 12 and 15. As mentioned above, the present invention is not limited to this sequence.

Each oligonucleotide (1, 3, 5) may comprise both natural and / or non-normal nucleotides. Thus, in one embodiment, the oligonucleotides (1, 3, 5) may comprise both natural and / or non-normal nucleotides. In another embodiment, the unnatural nucleotide is selected from the group consisting of PNA, LNA, xylo-LNA-, phosphorothioate-, 2'-methoxy-, 2'-methoxyethoxy - (2'-methoxyethoxy-), morpholino- and phosphoramidate- containing molecules or analogs thereof. For example, the advantage of non-natural nucleotides can be biostable, as they can be less degraded by nuclease. However, oligonucleotides may also consist of DNA and / or RNA. Thus, in other embodiments, the oligonucleotide can be composed of DNA or RNA, preferably native nucleic acid, such as DNA.

The term "oligonucleotide "," nucleic acid ", "nucleic acid molecule ", or" nucleic acid sequence ", as used herein, refers to oligomers or ribonucleic acid polymers or deoxyribonucleic acid (DNA) mimics / mimetics. The term is intended to encompass molecules composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) phosphodiester bond linkages with shared internucleoside (backbone) Naturally occurring nucleobases, molecules comprised of a sugar and a shared internal nucleoside (backbone) phosphodiester linkage, or a combination thereof. Such modified or substituted nucleic acids are often preferred over native forms due to, for example, improved cellular uptake, improved affinity for nucleic acid targets and increased stability in the presence of nuclease and other enzymes, Quot; nucleic acid analogues "or" nucleic acid mimics ". Preferred examples of nucleic acid mimetic / mimetic are peptide nucleic acid (PNA-), Locked Nucleic Acid (LNA-), xylo-LNA- (xylo-LNA-), phosphorothioate- (2'-methoxyethoxy-, 2'-methoxyethoxy-, morpholino- and phosphoramidate- (2'-methoxyethoxy-) phosphoramidate-) containing molecule or an analogue thereof.

A nucleic acid, nucleic acid molecule or nucleic acid sequence is, for example, composed entirely of a deoxyribonucleotide, a whole ribonucleotide, a nucleic acid mimetic or an analogue, or a chimeric mixture thereof. Monomers are usually linked by internal nucleotide phosphodiester bond links. Nucleic acids generally vary in size from small monomer units, such as 5-40, to thousands of monomer units, on an oligonucleotide basis. Whenever a nucleic acid or nucleic acid sequence appears, the nucleotide is 5 'to 3' from left to right, unless otherwise specified, "A" means deoxyadenosine, "C" means deoxythiidine refers to deoxycytidine, "G" refers to deoxyguanosine, and "T" refers to thymidine.

Non-natural  Nucleotides ( Unnatural nucleotides )

As used herein, "non-natural nucleotide" or "nucleic acid analog" or "artificial nucleotide" is understood to mean a structural analogue of DNA or RNA designed to hybridize to the complementary nucleic acid sequence (1). Through modification of the internal nucleotide linkage (s), sugar and / or nucleobase, the nucleic acid analog can obtain some or all of the following desirable characteristics: 1) optimized hybridization specificity or affinity, 2) nuclease resistance, 3) chemical stability, 4) solubility, 5) membrane-permeability, and 6) ease or low cost of synthesis and purification. Examples of nucleic acid analogs include peptide nucleic acids (PNA), locked nucleic acids (LNA), 2'-O-methyl nucleic acids, 2'-fluoro nucleic acids But are not limited to, 2'-fluoro nucleic acids, phosphorothioates, and metal phosphonates.

To hold  domain( Toehold regions )

In this context, a "tohod region" refers to two complementary oligonucleotide regions capable of forming a Watson-Crick base pairing, or the tohod region comprises a single strand sequence located at an oligonucleotide . Thus, when used in reference to a single stranded oligonucleotide, the term " tohod region " refers to a single stranded tohold region, while the term " (When hybridized to each other), it is understood to refer to the double stranded region. In this context, the complementary sequences of the double-stranded to-hold region can be distinguished as X and X ', respectively.

The tohold region may have other locations. In one embodiment, the first (single strand) toothed region 8 'of the third oligonucleotide 5 and the second (single strand) second toothed region 9' As shown in FIG. The position of the single-stranded to-hold regions 8 'and 9' on the opposite side of the split-point moving region can speed up the analysis.

In order to minimize cross-hybridization between different tohaled regions, the two tohred regions [8, 8 '] and [9, 9'] may have different sequences. Thus, in one embodiment, the first to fourth region 7 of the first oligonucleotide 1 and the second toe region 8 of the second oligonucleotide 1 are similar. In this context, the term non-relative is thus to have a different sequence and is therefore understood not to be 100% identical.

.

In addition, the two single stranded tohod regions may have different lengths from each other. Hybridization shifts in this way can be more easily recognized. Thus, in yet another embodiment, the length of the first toothed region 8 can be from 1 to 8 nucleotides, 1-6 nucleotides, 1-4 nucleotides, 2-10 nucleotides, 3-10 nucleotides, 4-10 nucleotides, 5- Such as 10 nucleotides, 7-10 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides or 6 nucleotides.

The term "hybridization" or "annealing ", as used herein, refers to the formation of a double stranded structure, such as one stranded nucleotide of a double stranded structure forms a specific Watson- Crick base pairing with a nucleotide of the opposite strand Refers to the association of single-stranded nucleotides. The term also encompasses pairs of nucleoside analogs such as deoxyinosine, nucleosides that are 2-aminopurine bases, and analogs thereof, which are used as oligonucleotides in accordance with the present invention Can be introduced.

In another embodiment, the first to fourth region 8 of the first oligonucleotide 1 and the first toe region 8 'of the third oligonucleotide 5 are 2-10, 3-10, 4- 10, 5-10, 2-8, 2-7, 2-6, 2-5, 2-4.

In another embodiment, the first toothed region 8 of the first oligonucleotide 1 has a first to fourth region 8 'of the third oligonucleotide 5 that is 70-100% complementary, 75-100 % Complementary, 80-100% complementary, 85-100% complementary, 90-100% complementary, 95-100% complementary, 97-100% complementary, 99-100% complementary or 100% complementary Likewise at least 70% complementary.

In another embodiment, the length of the second toothed region 9, 9 'is 1-8 nucleotides, 1-6 nucleotides, 1-4 nucleotides, 2-10 nucleotides, 3-10 nucleotides, 4-10 nucleotides, 5 10 nucleotides, such as 10 nucleotides, 7 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, or 6 nucleotides.

In one embodiment, the second toe region 9 of the second oligonucleotide 3 and the second toe region 9 'of the third oligonucleotide 5 are 2-10, 3-10, 4- 10, 5-10, 2-8, 2-7, 2-6, 2-5, 2-4.

Branch point movement area ( Branch migration region )

In this context, "splitting point shift" means that the hybridization equilibrium between the first and third oligonucleotides is directed toward hybridization between the second and third oligonucleotides, and conversely, through the hybridization of the two to- . Figure 2 shows how break point movement and how the equilibrium situation changes with the coupling of the analyte.

Because the branch point translocation region is a complementary region, it enables branch point transfer between the first and third oligonucleotides and between the second and third oligonucleotides. Thus, in one embodiment, the length of the branch point transition regions 7, 7 'is 4-30, 5-30, 7-30, 9-30, 11-30, 15-30, 20-30, 25-30 , 3-30 nucleotides, such as 3-25, 3-20, 3-15, 3-11, 3-9, 3-7, 3-5 or 3-4 nucleotides. Preferred lengths are applicable to different temperatures and specific sequences. In another embodiment, the branch point shifting region 7 of the second oligonucleotide 3 and the branch point shifting region 7 'of the third oligonucleotide 5 are 4-30, 5-30, 7-30, 9-30, 11-30, 15-30, 20-30, 25-30, 3-25, 3-20, 3-15, 3-11, 3-9, 3-7, 3-5 or 3- 4 < / RTI > complementary nucleotides, such as 4 complementary nucleotides.

In another embodiment, the breakpoint region 7 'of the second oligonucleotide 3 and the breakpoint region 7' of the third oligonucleotide 5 are 70-100% complementary, 75-100% complementary Such as 100-100% complementary, 85-100% complementary, 90-100% complementary, 95-100% complementary, 97-100% complementary, 99-100% complementary, 100% complementary 70% complementary. In another embodiment, the junction point shifting region 7 of the first oligonucleotide 1 and the junction point shifting region 7 of the second oligonucleotide 3 are 4-30, 5-30, 7-30, 9 -30, 11-30, 15-30, 20-30, 25-30, 3-25, 3-20, 3-15, 3-11, 3-9, 3-7, 3-5 or 3-4 Lt; RTI ID = 0.0 > complementary < / RTI > nucleotides.

In another embodiment, the first and second oligonucleotides (1, 3) are 70-100% identical, 75-100% identical, 80 100-100% identical, 85-100% identical, 90-100% identical, 95-100% identical, 97-100% identical, 99-100% identical or 100% identical. Thus, the two splitting point regions 7 of the first or second oligonucleotide do not need to be completely identical.

In another embodiment, the branch point shifting region 7 of the first oligonucleotide 1 and the branch point shifting region 7 'of the third oligonucleotide 5 are 4-30, 5-30, 7-30, 9-30, 11-30, 15-30, 20-30, 25-30, 3-25, 3-20, 3-15, 3-11, 3-9, 3-7, 3-5 or 3- 4 < / RTI > complementary nucleotides. In yet another embodiment, the first oligonucleotide (1) and the branch point shifting region (7 ') of the third oligonucleotide (5) are 70-100% complementary, 75-100% complementary, 80-100% complementary At least 70% complementary, such as 85-100% complementary, 90-100% complementary, 95-100% complementary, 97-100% complementary, 99-100% complementary or 100% complementary.

로 계산할 수 있으며, 여기서 N_dif는 정렬하였을 때 두 서열의 비-동일 잔기의 총 수이며, 여기서 N_ref는 한 서열의 잔기의 수이다. 따라서, DNA 서열 AGTCAGTC는 서열 AATCAATC(N_dif=2 및 N_ref=8)와 75%의 서열 동일을 갖는다. 갭은 특정 잔기의 비-동일로서 계산되어, 즉, DNA 서열 AGTGTC은 서열 AGTCAGTC(N_dif=2 및 N_ref=8)와 75%의 서열 동일을 갖는다.The term " sequence identical "indicates quantitative analysis of the degree of homology between two amino acid sequences of the same length or between two nucleic acids. If the two sequences that can be compared are not of the same length, they are aligned to provide the best possible fit, either by insertion of a gap or alternatively by permitting cleavage at the ends of the polypeptide sequence or nucleotide sequence. The sequence is the same

, Where N _dif is the total number of non-identical residues of the two sequences when aligned, where N _ref is the number of residues in one sequence. Thus, the DNA sequence AGTCAGTC has 75% sequence identity with the sequence AATCAATC (N _dif = 2 and N _ref = 8). The gap is calculated as the non-identical of the specific residues, i.e., the DNA sequence AGTGTC has 75% sequence identity with the sequence AGTCAGTC (N _dif = 2 and N _ref = 8).

Sequence identity between one or more sequences of all polypeptides or amino acids in embodiments of the invention is performed by clustalW software (www.ebi.ac.uk/clustalW/index.html) using default settings of the program Based on the alignment of each sequence. For embodiments of the invention based on nucleotides, the sequence identity between one or more sequences is therefore based on alignment using the default setting clustalW software. These settings for the nucleotide sequence alignment are: Alignment = 3D Whole, Gap Open 10.00, Gap Ext. 0.20, Gap separation Dist. 4, DNA weight matrix: identity (IUB).

It is understood that the degree of complementarity can be measured in a manner similar to using a complementary sequence of one oligonucleotide.

In one embodiment, the hybridization between the branching point shifting region 7 and the branching point shifting region 7 'comprises one or more hairpins in the branching point shifting region, such as 1-5 hairpin, 1-3 hairpin, 1-2 hairpin or 1 hairpin . This is not to be bound by theory, but with two adjacent hairpins in the middle of S, the whole complex of AS has a holiday junction, expanding a specific 3D structure in the presence of Mg2 +, and thus possibly competing with A, , Resulting in a larger steric hindrance to the protein-bound B strands (Fig. 20). In one embodiment, the at least one hairpin has a length of 1-20 nucleotides such as 3-20 nucleotides, 5-20 nucleotides, 10-20 nucleotides, 3- 15 nucleotides, 3-10 nucleotides and also 5- 15 nucleotides.

Combination Moiety ( Binding moiety )

The binding moieties (or moieties) according to the present invention allow this assay to detect an analyte that does not form a Watson-Crick base pair. Similarly, it enables the detection of an analyte that does not bind to DNA or RNA, such as a DNA binding protein.

The one or more binding moieties may in principle be located in one of the three oligonucleotides. In one embodiment, at least one covalently bonded moiety (4) is located in the first oligonucleotide (1). In other embodiments, at least one covalently bonded moiety (4) is located at the second oligonucleotide (3). In a third embodiment, at least one covalently bonded moiety (4) is located at the third oligonucleotide (5). In one embodiment, the binding detection system comprises 1-5 binding moieties as 1-4, 1-3, 1-2, 1, 2-5, or 3-5.

One or more bond moieties may be located in different regions. In one embodiment, the at least one covalently bonded moiety (4) is attached to the first oligonucleotide (1), the second oligonucleotide (3) or the third oligonucleotide (5) Lt; / RTI >

One or more of the covalently bonded binding moieties (4) in one embodiment may be selected from the group consisting of organic molecules, antibodies, antigens, aptamers, biotin And haptens. In another embodiment, the antigen is a protein antigen, a peptide antigen, or a sugar antigen. In some cases, covalent linkage is not necessary because some analyte can be directly conjugated to one or several nucleotides. Such analytes include DNA-binding proteins, some ions (Figure 19b) or intercalators, and small molecules such as melanin (Figure 19a). In one embodiment, the bonding moiety also has a sandwich structure in that the first covalently bonded moiety acts as a handle of a second bonding moiety that can be covalently or non-covalently bonded to the first moiety . Thus, the second binding moiety may have a dual function: I) binding to the first moiety and II) binding site for the analyte. An aptamer that forms one or more portions of an oligonucleotide and, when it is bound to the analyte, a separate covalently bonded binding moiety may not be necessary.

Small organic molecules are preferred as bonding moieties. In one embodiment, the organic molecule may be selected from the group consisting of 150-1200 Da, 150-1000 Da, 150-800 Da, 150-600 Da, 150-400 Da, 150-300 Da, 300-1500 Da, Da has a molecular weight range of 150-1500 Da (daltons) such as 800-1500 Da, 1000-1500 Da or 1200-1500 Da. Small organic molecules in the present invention are also preferred as analytes. In a more specific embodiment, the at least one covalently bonded moiety (4) is selected from the group consisting of vitamin D, folate, enrofloxacin, digoxigenin. Thus, examples of the organic molecule and / or the covalent bond moiety according to the present invention include:

Toxins : Staphylococcal enterotoxin B (SEB), Staphylococcal enterotoxin A (SEA), Domoic acid (DA), Aflatoxin (AFB1, AFG1, AFB2 , AFG2, AFM1), deoxynivalenol, ochratoxin A (OTA).

Drugs: Oral anticoagulant warfarin, insulin.

Insecticides : Atrazine, simazine, chlorpyrifos, carbaryl, dichlorodiphenyltrichloroethane (DDT), 2,4-dichlorophenoxyacetic acid (2,4-dichlorophenoxyacetic acid -dichlorophenoxyacetic < / RTI > acid (2,4-D)).

Detected in different environments : 2-hydroxybiphenyl (HBP), benzo [a] pyrene (BaP). Phenol (bisphenol A, atrazine, polychlorinated biphenyls, 3,7,8-TCDD), melanin and related compounds.

Veterinary drugs / antibiotics: penicillins and cephalosporins, chloramphenicol and chloramphenicol glucuronide, fenicol antibiotic residues, tetracycline, tyrosine tylosin, erythromycin, sulfonamide antibiotics.

Chemical contaminants: 4-nonylphenol in shellfish, insulin-like growth factor-1 (IGF-1).

Vitamin: Vitamin B2 (riboflavin), vitamin B5 (patotenic acid), vitamin B8 (biotin), vitamin B9 (folic acid), vitamin B12 (cobalamin).

Hormones: progesterone, human chorionic gonadotropin hormone (hCG), 17β-estradiol, R-fetoprotein (AFP), testosterone, 19-norestosterone 19-nortestosterone, methyltestosterone, boldenone and methylboldenone.

The explosives are 2,4,6-trinitrotoluene (TNT), 2,4,6-trinitrophenol (TNP), 1,3,5- (TNB), triacetonetriperoxide (TATP), hexamethylene triperoxide diamine (HMTD), pentaerythritol tetranitrate (tetranitrate) (PETN)), cyclotrimethylenetrinitramine (RDX).

It is understood that the compound list can also be an analyte to be detected in accordance with the present invention.

Antibodies and other proteins Analyzes:

Diagnostic antibodies: Mycoplasma hyopneumoniae antibodies, Classical swine fever virus (CSFV) antibodies.

Antibodies against viral pathogens: antibodies against hepatitis G, antibodies against hepatitis B virus (hHBV), antibodies against herpes simplex virus type 1 and type 2 (HSV-1, HSV-2) Antibody to Epstein-Barr virus (anti-EBNA), antibody to human respiratory syncytial virus (RSV), anti-adenovirus antibody.

Agent -directed antibody: an antibody to an insulin granule macrophage colony stimulating factor (GM-CSF), an antibody to a recombinant or non-recombinant protein or antibody drug.

Protein: casein, immunoglobulin G, folate-binding protein, lactoferrin, and lactoperoxidase.

Allergen (allergen) or allergy markers: peanut allergen, Con albumin of pasta / troponin myosin (Conalbumin / Tropomyosin), sesame protein, troponin myosin, immunoglobulin E (IgE) antibodies, histamine (α- imidazole ethylamine (α -imidazole ethylamine).

Cancer markers : prostate-specific antigen (PSA), PSA-ACT complex (α1-antichymotrypsin, carbohydrate antigen (CA 19-9)), protein vascular endothelial growth factor vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), carcinoembryonic antigen (CEA), fibronectin.

Other markers: troponin (troponin (cTn I)), antibodies, wherein for the glucose-6-phosphate (glucose 6-phosphate isomerase (GPI )) - glutamic acid decarboxylase enzyme antibody (anti-glutamic acid decarboxylase (GAD ) antibodies) , C-reactive protein (CRP), cystatin C, hepatitis B surface antigen (HBsAg).

The binding moiety may have its binding partner bound to the binding moiety. The term "binding partner" in this context is understood to mean a molecule capable of non-covalent association with the binding moiety. Thus, in one embodiment, the binding partner is non-covalently associated with the binding moiety. Non-limiting examples of a "binding moiety" - "binding partner" pair are antibody-antigens, streptavidin-biotin, folate receptor-folate. Thus, in one embodiment, the binding moiety has its binding partner bound to the binding moiety.

Depending on the portion of the type of analyte to be detected, three strategies can be applied to the detection system of the present invention. This strategy is described below, for example with reference to detection of protein or antibody (strategy 1) or small molecule analyte (strategies 2 and 3).

Strategy 1: direct analysis

In one embodiment (Strategy 1) the protein or antibody is the analyte to be detected (Figure 5, Strategy 1). In using this strategy, binding of the protein or antibody (analyte) to the binding moiety shifts the hybridization equilibrium and can be detected subsequently. This strategy is described in more detail in Figures 3-5 and corresponding Examples 1 and 2.

Strategy 2: Low Piracy Analysis

In another embodiment (strategy 2), a sample containing (or suspected of containing) a small molecule analyte (target molecule) is first mixed with a binding partner of the binding moiety (e. G., A protein such as an antibody) The mixture is then added to the remainder of the detection system (Figure 5, strategy 2). If the sample contains a small molecule of target (analyte), it is a protein that occupies the binding site of the protein (binding partner) and thereby interferes with the hybridization equilibrium through interaction with the same covalently bound small molecule bound to oligonucleotide B Resulting in little or no coupling capability. Thus, the presence of the analyte in the sample can cause a change in the hybridization equilibrium between the oligonucleotides of the detection system. This is called low-pit analysis. Figure 4E shows the results of the assay for biotin detection.

Strategy 3: Competitive Analysis

In another embodiment (Strategy 3), the binding partner (protein) is first added to the assay, resulting in the same equilibrium shift as in Strategy 1, followed by an unknown sample that is supposed to contain the analyte (target molecule) Lt; / RTI > Once the free analyte molecule is present, it can substitute for the covalently bonded binding moiety that binds to the binding partner (protein) on the oligonucleotide B that binds to the protein, thus resulting in the hybridization equilibrium (Figure 5, Strategy 3) Change. Therefore, this strategy is a competitive method. In both the pirate and competitive strategies, the signal changes more drastically compared to existing methods, and there are fewer target substances in the sample.

Analyte

Other classes of analyte can be detected by the present invention. The above-mentioned analyte is not DNA or RNA. Likewise, the analyte may not be an analyte that interacts with DNA, such as DNA or RNA binding proteins. An example of an analyte that does not form a Watson-Crick base pair but binds to DNA is a DNA binding protein, such as histone. In one embodiment, the analyte is a non-DNA and non-RNA binding analyte.

In one embodiment, the analyte can be selected from the group consisting of proteins, peptides, organic molecules, antibodies, antigens, sugars, lipids and haptens. In another embodiment, the analyte is a protein antigen, a peptide antigen, or a sugar antigen. In another embodiment, the analyte is at least one of 150-1200 Da, 150-1000 Da, 150-800 Da, 150-600 Da, 150-400 Da, 150-300 Da, 300-1500 Da, 400-1500 Da, Is an organic molecule having a molecular weight range of 150-1500 Da, such as 600-1500 Da, 800-1500 Da, 1000-1500 Da, or 1200-1500 Da. In certain embodiments, the analyte may be selected from the group consisting of antibiotics such as vitamins, toxins, allergens, drugs, cocaine, antibiotics such as enolactacin, insecticides, hormones, chemical contaminants, biomarkers. Thus, this is a more extensive list of analytes according to the present invention.

Signal system ( Signaling system )

A detection system according to the present invention includes a signaling system that provides a signal or a signal change when an analyte is detected. A signaling system is based on the principle that a signal is generated or promoted when an oligonucleotide comprising another part of the signaling system is hybridized. For example, as shown in Example 1, when hybridization of oligonucleotides 3 and 5 does not cause signal generation, oligonucleotides 1 (A) and 5 ( S) produces an increased signal. Thus, in one embodiment, the second oligonucleotide (3) comprises a first group (2), said first group forming a first part of the signaling system and a third oligonucleotide (5) (6), the second group forming a second part of the signaling system.

Likewise, the signaling system can be partitioned between the first and third oligonucleotides. Thus, in one embodiment, the first oligonucleotide (1) comprises a first group (2), said first group forming a first part of the signaling system and the third oligonucleotide (5) (6), the second group forming a second part of the signaling system.

The signaling system may also include a third portion that provides a different signal depending on the hybridized oligonucleotide, which means each of the oligonucleotides. Thus, in one embodiment,

- the first oligonucleotide (1) comprises a first group (2), said first group forming a first part of the signal system;

- the second oligonucleotide (3) comprises a third group (10), said third group (10) forming a third part of the signaling system;

- the third oligonucleotide (5) comprises a second group (6), said second group forming a second part of the signaling system.

This setup is shown in FIG. 4 and the corresponding embodiment.

Other types of signaling systems may be used. Thus, in one embodiment, the signal system formed by the first group 2 and the second group 6 comprises a quencher-fluorophore signal system, a fluorescence-quencher signal system , A FRET signaling system, a DNA peroxidase catalysis signaling system, or a quencher and singlet oxygen sensitizer. Signal systems can use fluorescent nanoparticles, particularly in the case of FRET or quencher systems. The example section provides examples of different setups that may be used in accordance with the present invention. In another embodiment

I. The first group (2) forming the first part of the signaling system is a quencher, the second group (6) forming the second part of the signaling system is a fluorophore, or

Ⅱ. The first group 2 forming the first part of the signal system is a fluorescent stage and the second group 6 forming the second part of the signal system is a quencher,

Ⅲ. The first group 2 forming the first part of the signaling system is a FRET and the second group 6 forming the second part of the signaling system is a FRET acceptor,

IV. The first group 2 forming the first part of the signaling system is a FRET acceptor and the second group 6 forming the second part of the signaling system is a FRET donor,

Ⅴ. The first group 2 forming the first part of the signal system is the first half of the DNA peroxidase signaling system and the second group 6 forming the second part of the signaling system is the DNA peroxidase The second half of the signaling system.

When using a DNA peroxidase signaling system, the detection system may further comprise components. Thus, in another embodiment, the detection system comprises hemin and / or ABTS ^{2 -} and / or H ₂ O ₂ and / or luminol. Colorimetric methods, such as gold nanoparticles (AuNP) or quantum dots (QD), can be integrated into the assay according to the present invention.

Other parts of the signal system can be covalently linked to other regions of the oligonucleotide. Thus, in one embodiment, the first portion of the signaling system and / or the second portion of the signaling system and / or the third portion of the signaling system are covalently coupled to portions of the oligonucleotide's branching point shifting regions 7, 7 ' Paired.

In another embodiment, the first portion of the signal system and / or the second portion of the signal system and / or the third portion of the signal system are covalently coupled to a portion of the tohod region 7, 8 of the oligonucleotide .

Those skilled in the art are aware of how to design a FRET pair in which, for example, a signal is generated or increased when the FRET pair is in close proximity. Similarly, those skilled in the art are aware of how to design a pair of fluorescent end-quenching moieties where the signal disappears or weakens when the fluorescent end-quencher pair is in proximity. Example 3 and the figures show how DNA peroxidase assays can be designed to facilitate signal generation when oligonucleotides each containing a portion of the DNA peroxidase signal system are brought into proximity. One advantage of the DNA peroxidase signaling system is that it constitutes an amplification reaction that allows detection of very small amounts of the analyte. Another advantage is that oligonucleotides can be synthesized more easily because less modification is required. In all of the above examples, the other pairs are brought closer because of the hybridization of the two oligonucleotides comprising each part of the signal system. As described above, since this assay is based on an equilibrium reaction, therefore, for example, the signal may be strong or weak depending on the change in hybridization equilibrium by binding or separation with the covalently bonded moiety and the binding partner .

In certain embodiments, the analyte can be a nucleic acid molecule, such as DNA or RNA, and the signaling system is conditionally a DNA peroxidase signaling system.

In other specific embodiments, the analyte may be a structurally related compound capable of interacting with melanin, and either one, two, or three thymine bases of oligonucleotides (1), (3) and / or (5).

share Combined  Oligonucleotide

In some cases, it may be a disadvantage that the detection system consists of multiple oligonucleotides, since this can increase the time before reaching the hybridization equilibrium. Oligonucleotides according to the present invention may also be covalently bonded to each other, meaning that the detection system consists of only one or two individual oligonucleotides instead of three oligonucleotides. This principle is shown in Experimental Example 12 and corresponding Figures 17 and 18. Thus, in one embodiment, the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide are covalently bonded.

Thus, in one embodiment, the first oligonucleotide (1) is covalently bound to the second oligonucleotide (3). In other embodiments, the third oligonucleotide (5) is covalently bound to the first oligonucleotide (1). In another embodiment, the second oligonucleotide (3) is covalently bonded to the third oligonucleotide (5). In another embodiment, the first oligonucleotide (1) is covalently bound to a second oligonucleotide (3) and the second oligonucleotide (3) is covalently bonded to a third oligonucleotide. In another embodiment, the 3'-end of the first oligonucleotide (1) is covalently bonded to the 5'-end of the second oligonucleotide (3). In another embodiment, the 3'-end of the second oligonucleotide (3) is covalently bonded to the 5'-end of the third oligonucleotide.

When two or more oligonucleotides are covalently bonded to each other, it is understood that the binding system according to the present invention continuously contains first, second and third oligonucleotides.

In certain embodiments, the analyte can be a nucleic acid molecule, such as DNA or RNA, and conditionally two or more oligonucleotides are covalently linked as described above.

In another embodiment, the oligonucleotide is covalently linked by a linker. In other further embodiments the linker is selected from the group consisting of phosphodiester bond linkages, nucleotides such as 1-20, such as 1- 10, 1-5, 3- 10, oligonucleotides, peptides, C A linker, a PEG-linker, a disulfide linker, and a sulfide linker. Those skilled in the art will be able to find other suitable connectors.

Partial Kit

The detection system is provided in the form of a kit of parts. Accordingly, aspects of the invention relate to a kit of parts comprising an analyte detection system in accordance with the present invention.

In another aspect the present invention relates to a kit of parts comprising a first oligonucleotide 1, a second oligonucleotide 3, and a third oligonucleotide 5 according to the invention.

The kit may further comprise components. Thus, in one embodiment, the kit further comprises hemin and / or ABTS ^{2 -} and / or H ₂ O ₂ and / or luminol. These components are the kit parts when using DNA peroxidase assays. As described above, it may be advantageous to include a binding partner of the binding moiety in the kit. Thus, in another embodiment, the kit comprises a binding partner of a binding moiety. In another embodiment, the binding partner is non-covalently paired with one or more binding moieties. In yet another embodiment, the binding partner is in a separate compartment of the kit rather than an oligonucleotide comprising a covalently linked binding moiety.

Of the sample Analyte  Method for presence or level detection

The present invention also provides a method for detecting an analyte in a sample. An aspect of the present invention, therefore,

comprising the steps of: a) providing a sample containing or suspected of containing an analyte of interest;

b) providing an analyte detection system in accordance with the present invention;

c) incubating the sample in the analyte detection system;

d) comparing the reference level with the detected level of the analyte; And

e) measuring the presence or level of the analyte in the sample;

And to methods for detecting the presence or level of other analytes and DNA and RNA of the sample.

It is understood that the presence or absence of the analyte in the sample may be unknown, or that the sample is known to be included. In the first case the quantification may be the target, but in the second case it may be sufficient to just detect the presence.

In another embodiment, DNA and RNA and other analytes are not nucleic acid molecules.

Preferably, the incubation step is continued until the hybridization equilibrium is reached. Thus, in one embodiment, the incubation step c) continues until equilibrium is reached. However, this method can also be verified in real time. The hybridization equilibrium can vary depending on the binding of the analyte to the binding moiety. Thus, in one embodiment, the hybridization equilibrium between the first oligonucleotide (1) and the third oligonucleotide (5) and between the second oligonucleotide (3) and the third oligonucleotide (5) .

If the binding partner of the binding moiety is included in this assay, the hybridization equilibrium is altered in a different way. Thus, in other embodiments, the hybridization equilibrium between the first oligonucleotide (1) and the third oligonucleotide (5) and between the second oligonucleotide (3) and the third oligonucleotide (5) ) According to the separation of the binding partner.

Binding partners of binding moieties can be included in this assay at different points in time. In another embodiment, the binding partner of the binding moiety is incubated with the sample before the sample is incubated with the oligonucleotide of the detection system. In another embodiment, after the sample is incubated with the oligonucleotide of the detection system, the binding partner of the binding moiety is incubated with the sample. In other embodiments, the binding partner of the binding moiety is incubated with the sample prior to incubation with the oligonucleotide of the detection system. Depending on the binding affinity, the solutions may each be most suitable. See Figure 13 for further details.

In other embodiments, separation of the binding partner from the binding moiety is caused by binding of the binding partner to the analyte.

The incubation time may vary from assay to assay depending on the sample type, the analyte and the exact oligonucleotide used. Thus, in one embodiment, the incubation step c) is performed at a temperature of between 1 minute and 12 hours, 1 minute -6 hours, 1 minute -2 hours, 1-60 minutes, 1-30 minutes, 1-15 minutes, 1-5 minutes 5- 60 minutes, 10-60 minutes, 15-60 minutes, 30-60 minutes, 1-6 hours, 2-6 hours, 4-6 hours.

The advantage of this method is that the temperature need not change during the analysis. Thus, in other embodiments, the method is performed under isothermal conditions. This means that this method is performed using only a heating chamber or a heating plate. Likewise, this assay can be carried out in situ at room temperature. This method can be analyzed later using the necessary equipment. If a DNA peroxidase signaling system is used, the results can be determined by visual inspection. In another embodiment, the process is performed at a temperature of 10-50 ° C, 20-50 ° C, 25-50 ° C, 30-50 ° C, 35-50 ° C, 40-50 ° C, 4-40 ° C, 4-35 ° C, 4-30 Deg.] C, 4-25 [deg.] C, or 4-20 [deg.] C. In another embodiment, the method is performed under isothermal conditions.

Sample

Samples can be from a variety of sources. In one embodiment, the sample is a sample obtained from an environment, such as a water sample. In another embodiment, the sample is a biological sample. In another embodiment, the sample is food, or plastic. Plastics can be tested for the presence of softeners that can be toxic.

In another embodiment, the biological sample is obtained from a mammal such as a human. In other embodiments, the biological sample is a blood sample, such as serum or plasma, a urine sample, a feces sample, a biopsy sample, or a saliva sample. To provide optimal conditions for this assay, the sample may be purified or substantially purified prior to use in the assay according to the invention. Thus, in another embodiment, the sample is a purified sample. The advantage of the purified sample is that the reaction conditions are much easier to control.

This assay can be read by a variety of methods. Thus, in one embodiment, the presence or level of the analyte can be determined by visual inspection, absorbance, spectroscopy, spectroscopy, fluorescence spectroscopy, electrochemistry, QCM, SPR, or microscopy.

To be able to determine the level or presence, the sample may need to be compared to the standard level. Thus, in another embodiment, the standard level is a predetermined value, a standard curve, or a negative control. Standard levels can be set based on various standards, such as the use of ROC curves often used in diagnostic tests.

The accuracy of the diagnostic test can be characterized by the Receiver Operating Characteristic curve ("ROC curve"). ROC is a plot of the true positive rate against the false positive rate for the other possible cutoff points of the diagnostic test. The ROC curve represents the relationship between sensitivity and specificity. This is because an increase in sensitivity can be accompanied by a decrease in specificity. The closer the curve is to the left axis and closer to the top edge of the ROC space, the more elaborate the test is. Conversely, the closer the curve is to the 45-degree diagonal of the ROC graph, the less elaborate the test. The area under the ROC represents the inspection accuracy. The accuracy of the test depends on how well the group to be examined is distinguished as to whether the disease in question exists and is non-existent. An area under the curve (referred to as "AUC") is a perfect test, while an area of 0.5 indicates a less useful test. Thus, the biomarkers and diagnostic methods of the present invention have an AUC greater than 0.50, a more preferred assay has an AUC greater than 0.60, and a more preferred assay has an AUC greater than 0.70.

Other useful measures of the utility of testing are positive predictive and negative predictive. The positive predictive value is a positive rate in a person determined to be positive. The negative predictive value is the ratio of the actual voice to the person who has been confirmed as the negative. Thus, a person skilled in the art can measure the standard level based on specially required criteria.

It should be noted that the implementations and drawings described in the context of an embodiment of the present invention are also applicable to other embodiments of the present invention. It should be noted that reference numerals have been used for illustrating the specification and claims to illustrate the invention with reference to the drawings, which are for illustration purposes only and are not to be construed as limiting the invention.

All patents and non-patent references cited in this application are hereby incorporated by reference in their entirety.

The present invention will now be described in more detail in the following non-limiting examples.

Example  One

Protein detection: Streptavidin (STV)

material

Three DNA strands were used in this experiment, designated as A, B, and S, respectively. While B has an internal biotin modification, A and S have an internal amine modification that is used as a handle for an Alexa fluorophore label. The sequence is given below (underlined indicates the to-hold region):

All oligonucleotides were purchased from DNA Technology A / S of Denmark. RP-HPLC purification was carried out in-house after direct synthesis.

Alexa Fluor 647 Succinimidyl Ester and Alexa Fluor 5 succinimidyl ester were purchased from Invitrogen.

All other reagents, including streptavidin, were purchased from Sigma-Aldrich.

Figure 3 shows how oligonucleotides can hybridize to one another.

Way

Fluorophore  join

Including some variations, the labeling procedure refers to the protocol provided by Invitrogen. More specifically, amine-modified DNA (16 μl, 100 μl) was mixed with phosphate buffer (10 μl, 0.4 M, pH 8.5) and then activated dye-ester (100 Mg, 80 nmol) was added. In this mixture, the final DNA concentration is about 40 μM, and the molar ratio of ester to amine is 50: 1. After overnight incubation at 28 ° C, the mixture was ethanol precipitated and then subjected to reverse-phase HPLC purification (5-40% MeCN in 0.1 M TEAA over 15 min) on an Agilent 1200 Series. At 260 nm to give the sample corresponding to the maximum absorption peak and freeze-dried, again in 200 ㎕ H ₂ 0 - dissolved. The final concentration before use was determined with a NanoDrop 1000 spectrophotometer.

Structure of analytical method and its STV detection function

In a typical assay, 1: 1: 1 exact stoichiometric ratio strand of (stoichiometric ratio) of A, B and STV as S and the target ^{protein, 1x [TAE-Mg 2 +} ] buffer (40 mM Tris-HAc (pH 8 ), ImM EDTA, 12.5 mM Mg (Ac) ₂ ). Typical final concentrations were 20 nM for each DNA component and 250 nM for STV. Excessive STV was used to ensure primary coupling, but this is not necessary for practical detection. Kinetic data indicated that the system was sufficient for 3 hours to reach equilibrium (data not shown), but for convenience, the mixture was incubated overnight at room temperature (RT).

Spectral type optical system  by FRET  Measure

lx of the mixture between 70 ㎕ analyze different samples were pipetted in [TAE-Mg ^{2 +]} The quartz cuvette (quartz cuvette) and washed with two times. Fluorescence measurements were made using a scanning spectrophotometer (Fluoro-Max-3, HORIBA Jobin Yvon Inc.). Excitation for Alexa555 was performed at 530 nm. The FRET efficiency was calculated as E = I _A / (I _A + I _D ), where I _A is the acceptor peak fluorescence intensity (667 nm for Alexa 647), I _D is the donor peak fluorescence intensity 566 nm for Alexa555).

Gel experiment and Typhoon scanning

Each 15 μl sample used for the bulk FRET measurement was mixed with 3 μl of 6 × Gel Loading Dye (NEB) and subjected to 6% non-denaturing polyacrylamide gel electrophoresis (PAGE) gel acrylamide / bis acrylamide (acrylamide / bisacrylamide); 19: . after loading the wells of 1) was eluted from the refrigerator (70 V, 1.5 h) lx TBE-Mg 2 + ([Mg 2 +] = 12.5 mM) Was used to prepare both the gel itself and running buffer (running buffer).

After electrophoresis, the gel was scanned using Typhoon 9400 (Amersham Biosciences) in the fluorescence mode, without staining. A red laser (633 nm) and a 670 nm band-pass filter (transmitting light between 655 nm and 685 nm and centered on the transmission peak at 670 nm) excites and emits the Alexa647 dye emission were selected as the combination for each. With a 200 Mm pixel size, the typical scanning time for an 8.3 x 7.3 cm mini-gel was 3 minutes. The gel was then analyzed with ImageQuant TL 7.0 (GE Healthcare). Lane generation and band detection were performed manually.

result

When STV is absent, hybridization between the three strands of DNA can reach equilibrium, and when A is only 3 nt to hold, B has to be BS duplex more than AS duplex do. When there is an STV capable of binding to B through the biotin-STV interaction, only the bulkiness of the STV through the ability of B to substitute A to hybridize with S will interfere with the sensitive branch migration process The original equilibrium can be shifted, so that it should have less BS duplex and more AS duplex in the sample.

To provide a FRET signal representative of a population of AS duplexes in solution, two fluorophore pairs forming a FRET (Forster resonance energy transfer) pair were placed in each of A and S (see FIG. 3). Approximately twice the normalized FRET efficiency appeared after addition of STV (Fig. 4a), and its widely diffused representative unfractionated fluorescence data also appeared (Fig. 4b). In addition, titration curves are generated and shown in Figure 4c, which shows quantitative detection and sensitivity possibilities of sub-nanomoles. More rationale is identified in non-denatured PAGE gels where each component in solution can be observed individually (Figure 4d). It is noteworthy that the selected excitation and emission wavelength confirms that the band intensity reflects only the amount of A. The results fully confirm the expectation that much less single-strand A (ssA) is left, while more AS duplexes are formed in the presence of STV (Fig. 4d, one-biotin).

In this embodiment, also two biotin is applied to one B strand. This is due to the fact that STV has tetravalent binding capacity and biotinylated B can wrap curv up STV by cross-site interaction (Fig. 4d insertion) will be. In this way, the tohod can not function at all and therefore even fewer BSs and more ASs are expected. In fact, the changes are more impressive after the addition of STV than the one-biotin system (Fig. 4c, two-biotin).

comment

In addition to the above results, the following were observed (data not shown):

1. Fluorescent end-quencher pair was attempted as an alternative reporter system instead of FRET. This also provided suitable results.

2. Two negative controls were examined: STV was added to the system without biotin, and another protein (thrombin or other antibody) was added to the biotinylated system. Neither shows a significant FRET change.

3. The sensitivity of this sensing method depends on the sensitivity of the instrument used to measure FRET levels. In the present invention carried out so far, STVs lower than 1 nM have been successfully detected as target proteins by using assays with a final concentration of 1 nM DNA.

conclusion

The inventors have successfully used biotin-STV interactions as a model system to indicate that the equilibrium-based assays based on the three DNA strands have high potential for the detection of specific target proteins as well as small molecules. Both bulk FRET data and typing-scanning gel experiments proved this design and the signal can be doubled (or half as much as this design) after adding STV.

Example  2:

Small molecule detection: Biotin

By applying the suppression strategy of the system described in Example 1 (FIG. 5; strategy 2), biotin can be detected. In this example, the biotin to be detected as the target was first mixed with a predetermined amount of STV, and then the entire mixture was added to the standard assay. In the presence of free biotin, all active binding sites of STV can be occupied, and thus can not affect DNA equilibrium. This signal-off detection method was tested using a concentration gradient of 14 nM DNA, 20 nM STV and biotin. The titration curve is also shown in Figure 4e. The molar ratio between biotin and STV is in agreement with the quaternary nature of STV.

Way

Prior to using streptavidin addition with 14 nM DNA, the same assay setup as in Example 1 was used. Various concentrations of biotin were first premixed with streptavidin (20 nM). After incubation for 3 hours at room temperature, the mixture was added to the general assay and incubated overnight at room temperature under dark conditions.

conclusion

In the presence of biotin as a target molecule, STV exhibits the same effect on equilibrium as before. If the sample contains biotin, this free biotin blocks the binding site of STV so that STV can not be possible in the assay. This signal-off detection method provides a smooth calibration curve for detecting biotin, and this result reflects a quadruple STV (Fig. 4E).

Example  3

Specificity of biotin detection assay

Without any labeled binding moiety, the control system can contain three DNA strands with two FRET pairs. Any addition of streptavidin, IgG, thrombin, or ATP to this system does not cause a change in the detectable signal (Fig. 6A), not only to prove the design, As evidence of good specificity.

The specificity was also checked by adding various targets into the biotin system described in Example 1, none of which exhibited a nonspecific detection effect (Fig. 6B). This assay was therefore validated by the addition of STV as a control system without ligand, and no FRET signal changes were observed.

Example  4

Detection of anti-digoxigenin (aD) in buffer

material

Three DNA strands were used in this experiment, designated as A, B, and S, respectively. While B has an internal digoxigenin modification, A and S have an internal amine modification that is used as a handle for an Alexa fluorophore label. The sequence is given below (underlined indicates the to-hold region):

 All oligonucleotides of A, B, and S were synthesized in-house with a MerAmade-12 oligonucleotide synthesizer from Bioautomation. Following synthesis, the DNA strand was purified with TOP-cartridge and ethanol precipitated.

Alexa Fluor 647 Succinimidyl Ester and Alexa Fluor 555 succinimidyl ester were purchased from Invitrogen.

5-Aminoallyl-dU phosphoramidite was purchased from Berry & Associates.

Anti-digoxigenin was purchased from Roche.

All other reagents were purchased from Sigma-Aldrich.

Figure 8A shows how oligonucleotides can hybridize to one another.

Way

Fluorophore  join( Fluorophore conjugation )

The same as in Example 1.

Digoxigenin  join( Digoxigenin conjugation )

Amine-modified DNA (100 μL, 50 μM) was added to activated digoxigenin ester (DIG-NHS) in DMF (100 μl, 150 nmol) and triethylamine (2 μl) . The final concentration of DNA in this mixture is 25 [mu] M, and the molar ratio of ester to amine is 30: 1. After incubation at 25 ° C for 1 hour, the mixture was ethanol precipitated and then subjected to reverse phase HPLC purification (5-40% MeCN in 0.1 M TEAA over 15 minutes) on an Agilent 1200 Series. At 260 nm to give the sample corresponding to the maximum absorption peak and freeze-dried, again in 200 ㎕ H ₂ 0 - dissolved. The final concentration before use was determined with a NanoDrop 1000 spectrophotometer.

Structure and objection of analytical methods aD  Detection function

Identical to Example 1, with aD instead of STV as the target protein and anti-digoxigenin with 2-fold excess (20 nM of each DNA component and 40 nM of aD).

Spectral type optical system  by FRET  Measure

The preparation of the sample and the FRET measurement are the same as in Example 1.

Gel experiment and Typhoon scanning

The same as in Example 1.

result

The obtained results are shown in Fig. FRET levels are observed that reflect a 30% increase in AS in the presence of aD. The aD titration at various concentrations is shown in Figure 8D. A complementary detection method of aD by electrophoresis is shown in Fig. 8E.

Example  5

Detection of anti-digoxigenin in human plasma

material

Three DNA strands were used in this study, named A, B, and S, respectively. While B has an internal digucigenin (DIG) modification, A and S have an internal amine modification that is used as a handle for an Alexa fluorophore label. The sequence is given below (underlined indicates the to-hold region):

 All oligonucleotides of A, B, and S were synthesized in-house with a MerAmade-12 oligonucleotide synthesizer from Bioautomation. Following synthesis, the DNA strand was purified with TOP-cartridge and ethanol precipitated.

Alexa Fluor 647 Succinimidyl Ester and Alexa Fluor 555 succinimidyl ester were purchased from Invitrogen.

5-Aminoallyl-dU phosphoramidite was purchased from Berry & Associates.

Human whole blood was donated from the blood bank of the Aarhus University Hospital in Skejby, Denmark.

All other reagents were purchased from Sigma-Aldrich.

Way

Fluorophore  join

The same as in Example 1.

Digoxigenin  join

The same as the fourth embodiment.

Human plasma preparation ( Human plasma preparation )

In the hospital collected from donors, human whole blood was buffered with EDTA, and plasma was separated from samples within 30 minutes of blood collection. This was carried out by centrifugation at 3000g, 15 minutes, 20 ° C. The upper layers that make up the plasma were carefully pipetted and immediately frozen with smaller aliquots.

Structure and objection of analytical methods aD  Detection function

The same as the fifth embodiment. However, prior to overnight incubation, the antibody of interest (aD) was added to the pre-mixed 15% v / v pure serum.

Spectral type optical system  by FRET  Measure

Sample preparation and FRET analysis are the same as in Example 4. However, the spectrum containing the human plasma is a value obtained by subtracting, using relative to the sample containing only human plasma in (10㎕ plasma of ^{60 ㎕ lx [TAE-Mg 2} +]) TAE-Mg buffer.

result

The results obtained from three independent experiments are shown in Fig. As can be observed, the AS ratios are the same before adding aD to buffers and plasma (Samples 1 and 2 in B). 2eg. As soon as the addition of aD, the Fret increase is gradually lowered only in the plasma (Sample 5) as compared to the buffer (Sample 3). But also in the detection results of digoxigenin (DIG) of buffer (Sample 4) and Plasma (Sample 6) according to Strategy 3; This is described in detail in Example 8.

Example  6

Detection of anti-digoxigenin (aD) in human saliva

material

Three DNA strands were used in this experiment, designated as A, B, and S, respectively. While B has an internal digucigenin (DIG) modification, A and S have an internal amine modification that is used as a handle for an Alexa fluorophore label. The sequence is given below (underlined indicates the to-hold region):

 All oligonucleotides of A, B, and S were synthesized in-house with a MerAmade-12 oligonucleotide synthesizer from Bioautomation. Following synthesis, the DNA strand was purified with a TOP-cartridge and ethanol precipitated.

Alexa Fluor 647 Succinimidyl Ester and Alexa Fluor 555 succinimidyl ester were purchased from Invitrogen.

5-Aminoallyl-dU phosphoramidite was purchased from Berry & Associates.

Anti-digoxigenin was purchased from Roche.

Human saliva was collected from fasting human donors for an hour.

All other reagents were purchased from Sigma-Aldrich.

Figure 10 shows how oligonucleotides can hybridize with one another.

Way

Fluorophore  join

The same as in Example 1.

Digoxigenin  join

The same as the fourth embodiment.

Human saliva preparation

Saliva was collected from a fasted male for 1 hour into a Falcon tube for 1 hour. The saliva was vortexed for 1 hour and then the saliva was centrifuged at 4 ° C and 10,000 g for 10 minutes. The liquid was separated from the solid and the saliva was filtered through a 100k Amicon Ultra-0.5 mL centrifuge filter.

Structure and objection of analytical methods aD  Detection function

The same as the fifth embodiment. But before incubation overnight, the antibody (aD) of interest in the sample was added to the pre-mixed 15% v / v filtered saliva.

Spectral type optical system  by FRET  Measure

The sample preparation and the FRET analysis are the same as in Example 7. However, the spectrum containing the human plasma is a value obtained by subtracting, using relative to the sample containing only human plasma in (10㎕ plasma of ^{60 ㎕ lx [TAE-Mg 2} +]) TAE-Mg buffer.

result

The FRET values reflecting the 28% population increase of AS in the presence of aD in human saliva as a result are comparable to Example 7 and are also shown in Fig.

Example  7

Detection of aD of various DIG modifications on DNA

material

Three DNA strands were used in this experiment, designated as A, B, and S, respectively. B has four internal digokigenin variants (not limited to four internal digokigenin variants), whereas A and S are internal amine modifications used as handles to Alexa fluorophore labels Respectively. The sequence is given below (underlined indicates the to-hold region):

 All oligonucleotides of A, B, and S were synthesized in-house with a MerAmade-12 oligonucleotide synthesizer from Bioautomation. Following synthesis, the DNA strand was purified with TOP-cartridge and ethanol precipitated.

Alexa Fluor 647 Succinimidyl Ester and Alexa Fluor 555 succinimidyl ester were purchased from Invitrogen.

5-Aminoallyl-dU phosphoramidite was purchased from Berry & Associates.

Anti-digoxigenin was purchased from Roche.

All other reagents were purchased from Sigma-Aldrich.

Figure 11 shows how oligonucleotides can hybridize to one another.

Way

Fluorophore  join

The same as in Example 1.

Digoxigenin  join

The same as the fourth embodiment.

Structure and objection of analytical methods aD  Detection function

The same as the first embodiment. Only aD instead of STV as the target protein, and anti-digoxigenin (20 nM of each DNA component and 60 nM of aD) exceeding 8-fold.

Spectral type optical system  by FRET  Measure

Sample preparation and FRET analysis are the same as in Example 1.

Gel experiment and Typhoon scanning

The same as in Example 1.

result

As a result, FRET levels indicating a 33% population increase of AS in the presence of aD can be compared with Example 4.

Example  8

Detection of digoxigenin (DIG) by strategies 2 and 3

Figure 12 shows how oligonucleotides can hybridize to one another.

material

Same as Examples 4, 5 and 6, and addition of digoxigenin was purchased from Sigma-Aldrich.

Way

Fluorophore  join

The same as in Example 1.

Digoxigenin  join

The same as the fourth embodiment.

Structure and objection of analytical methods DIG  Detection function

Samples are the same as Examples 4, 5 and 6, and free DIG is additionally added to various concentrations of samples for competitive assays.

Spectral type optical system  by FRET  Measure

Sample preparation and FRET measurements are the same as in Examples 7, 9 and 10, with or without the addition of human plasma and saliva, each signal modulation is performed.

result

This result is comparable to Examples 4, 5 and 6 and shows that when compared to the normal 30% increase in DIG in non-presence, a population increase of only 0.5% of AS in the presence of 320 nM DIG and a population increase of AS in the presence of 40 nM DIG The FRET value reflects a population increase of only 11%. For human plasma and saliva containing 40 nM DIG, the AS population increase is 5% and 2%, respectively, when DIG is compared to the 26% and 28% increases in the absence. The result is shown in Fig.

Experimental Example  9

Detection of vitamin D-binding protein (DBP) and vitamin D (VD).

material

Three DNA strands were used in this experiment, designated as A, B, and S, respectively. While B has an internal vitamin D modification, A and S have an internal amine modification that is used as a handle for an Alexa fluorophore label. The sequence is given below (underlined indicates the to-hold region):

 All oligonucleotides of A, B, and S were synthesized in-house with a MerAmade-12 oligonucleotide synthesizer from Bioautomation. Following synthesis, the DNA strand was purified with a TOP-cartridge and ethanol precipitated.

Alexa Fluor 647 Succinimidyl Ester and Alexa Fluor 555 succinimidyl ester were purchased from Invitrogen.

5-Aminoallyl-dU phosphoramidite was purchased from Berry & Associates.

Activated vitamin D ester was synthesized internally by a two - step method in cholecalciferol.

All other reagents were purchased from Sigma-Aldrich.

Figure 14 shows how oligonucleotides can hybridize to one another.

Way

Fluorophore  join

The same as in Example 1.

Vitamin D junction

Same as DIG-NHS as in Example 4, and the activated vitamin D ester replaces DIG-NHS.

Structure and objection of analytical methods DBP Detection function

The same as in Example 4, and DBP instead of aD as a target protein.

Spectral type optical system  by FRET  Measure

Sample preparation and FRET analysis are the same as in Example 1.

Gel experiment and Typhoon scanning

The same as in Example 1.

result

The results are comparable to those of Example 1, except that FRET values represent a 20% increase in AS in the presence of DBP and are therefore shown in FIG.

Moreover, low-cost and competitive strategies were used for vitamin D detection. Various concentrations of vitamin D were added to the analytical mixture prior to DBP protein addition (lowering strategy 2) or after (competitive strategy 3). Both methods were successful in detecting vitamin D as shown in the calibration curve of Fig.

Example  10

As an example of an aptamer system, ATP detection

material

Three DNA strands were used in this experiment, designated as A, B, and S, respectively. A and S have an internal amine modification that is used as a handle for a fluorescent (Alexa fluorophore) label. B does not have any additional modifications, but contains an ATP aptamer sequence at the 3 'terminus. The sequence is presented below (italic indicates tohred region; underlined indicates aptamer region):

All oligonucleotides were purchased from DNA Technology A / S of Denmark, and RP-HPLC purification was performed directly at the company after synthesis.

Alexa Fluor 647 Succinimidyl Ester and Alexa Fluor 555 succinimidyl ester were purchased from Invitrogen.

All other reagents were purchased from Sigma-Aldrich.

Way

Fluorophore  join

The same as in Example 1.

Structure and objection of analytical methods aF  Detection function

The concentration range of the ATP molecule instead of STV is the same as in the first embodiment.

Spectral type optical system  by FRET  Measure

The same as in Example 1.

result

The design of the present embodiment was conceived by a so-called structure-transfer aptamer, which typically undergoes target-directed conversion between a DNA duplex and an aptamer-target complex. Here, the portion of the aptamer (8 nt) is provided in the B-phase tohow, so it can hybridize with the S-phase toll, and A has a shorter tohold, resulting in a duplex dominance in solution. In the presence of the target (ATP), the aptamer-target bond is strong enough to competitively outcompete with the in-duplex hydrogen bond, leaving no functional tohold of B. Therefore, the AS duplex takes up the majority. A scheme is shown in Fig. 15A.

The titration curve was prepared in micromolar size (Fig. 15B). Up to 60% increase in FRET for AS duplex is observed in the presence of 1 mM ATP. The concentration of each DNA component used here was 20 nM.

Review

In the present embodiment, the inventor has combined a structure-switched aptamer design with a tohod exchange reaction system to perform target detection through an abdominal-target combination. Without amplification, changes in the FRET signal were already sufficiently significant for observation, and even quantification is possible. It should be noted that the aptamer system is functional as an aptamer region that is located at either the 5 'end or the 3' end.

Example  11

Streptavidin (STV) detection using DNA peroxidase signaling system

material

Three DNA strands were used in this experiment, named A, B and S, and only B was modified. The sequences are shown below (Italic indicates tophold region; underline indicates G-rich region for DNA peroxidase): < RTI ID = 0.0 >

All oligonucleotides were purchased from DNA Technology A / S of Denmark, and RP-HPLC purification was performed directly at the company after synthesis.

All other reagents were purchased from Sigma-Aldrich.

Way

Structure and objection of analytical methods STV   Detection function

In a typical assay, 1: 1: 1 correct stoichiometric ratio (stoichiometric ratio) strands A, B and S, and STV as the target ^{protein, 1x [TAE-Mg 2 +} ] buffer (40 mM Tris-HAc (pH 7 of ), ImM EDTA, 12.5 mM Mg (Ac) ₂ ). (3-ethylbenzothiazoline e-6-sulfonic acid) (2,2'-azino-bis (3-ethylbenzothiazoline e-6-sulfonic acid) (3-ethylbenzothiazoline-6-sulphonic acid) and H ₂ O ₂ (hydrogen peroxide) were added to the mixture, the mixture was incubated for 3 hours at room temperature. Typical final concentrations were 200 nM biotinylation the DNA, in 400 nM STV, was 2μΜ hemin and 2 mM of ABTS and H ₂ 0 _2.

On the nano drop  Absorbance measurement by

After incubation for 30 minutes at room temperature, 1.5 [mu] l of each assay mixture was pipetted onto a pedestal of a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific Inc.) and measured in UV-Vis mode. Absorbance at 420 nm was recorded.

conclusion

In this embodiment, the present inventors show an example of binding truncated DNAzyme at the ends of the amounts A and S at which the catalytic ability thereof can be restored when brought together by hybridization between A and B. The scheme of the two-biotin system is shown in FIG.

When STV is absent, B has a longer to hold, so S tends to associate with B. By the isolated part of the G-quadruplex, low peroxidase activity can be found. When STV is present, strand B loses its tohold contained in the protein surface, thus the formation of an AS duplex results in a full functional G-quadruple. Green appears after the addition of the requisite reactants such as H ₂ O ₂ , ABTS ^{2 -} and hemin, and the maximum absorbance at 420 nm can be recorded.

A significant change in absorbance can be seen in FIG. 16, and the difference can be visually distinguished. Note that changes in the one-biotin system therefore appear even though direct visualization is difficult to discern due to the stronger background.

conclusion

The present inventor used a new reporter system to provide visual signals in this assay. The advantage of this system is that it requires only pure DNA extension instead of covalent labeling, its catalyst and color information even providing direct, human detectable results. This method is thus applicable to a one-stranded system as described in embodiment 3. [

Example  12

One-stranded system in combination with DNA G-quadruple peroxidase for streptavidin (STV) detection

material

Only one strand of DNA is used in this experiment, named L. The internal amine group is a modified group at a specific position and is used to control the label. The sequence is shown below (italics indicate the to-hold region; the underline indicates the G-rich region for the DNA peroxidase):

All oligonucleotides were purchased from DNA Technology A / S of Denmark, and RP-HPLC purification was performed directly at the company after synthesis.

All other reagents, including streptavidin, were purchased from Sigma-Aldrich.

Figure 17 shows how an oligonucleotide can self-hybridize according to the presence of the analyte.

Location (nucleotides) 1-5 (2) is one fourth of G4-DNA (DNA peroxidase). Positions 6-7 (8) are the first toe. Position 8-17 (7 or A) is the first minute movement area. Position 18-27 (7 or B) is a second minute point moving region having the same sequence as the first minute point moving region, but giving small molecule strain. Positions 28-31 (9) are the second toe region. Positions 32-37 (11) are loop regions that provide flexibility. Positions 38-41 (9 ') are third tohold regions complementary to the second to-do region. Positions 42-51 (7 'or S) are the third minute point shift regions complementary to the first or second minute point shift regions. Positions 52-53 (8 ') are the fourth toe region complementary to the first toe. Region 54-66 (6) is the other three-quarters of the DNA peroxidase.

Way

Biotin junction

This procedure is the same as the fluorescent single conjugation process of Example 1, and is a biotin-NHS ester instead of a dye -NHS ester.

Structure and objection of analytical methods STV Detection function

In the general assay, STV was added to the lx [TAE-Mg2 +] buffer (40 mM Tris-HAc (pH 7), 1 mM EDTA, 12.5 mM Mg (Ac) 2) as the strand L and the target protein. The mixture was diluted with hemin (Ferriprotoporphyrin IX chloride), ABTS (2,2'-azino-bis (3-ethylbenzothiazoline e-6-sulfonic acid) (RT) for 3 hours prior to the addition of 200 nM of biotinylated DNA, 400 nM of STV, 2 [mu] M of hemin and 2 mM of 3-ethylbenzothiazoline-6-sulphonic acid It was ABTS and H ₂ 0 _2.

Nano drop  Measurement of absorbance by spectrophotometer

After incubation for 1 hour at room temperature, 1.5 [mu] l of each assay mixture was pipetted onto a nano drop 1000 spectrophotometer (Thermo Fisher Scientific Inc.) pedestal and measured in the UV-Vis mode. Absorbance at 420 nm was recorded.

result

The concept of this design is to simply mix all three oligonucleotides with a single long DNA strand, to avoid stoichiometry problems due to intramolecular reactions, and to have the potential to speed up the kinetics of strand displacement. To avoid the difficulties of making three transformations on one DNA strand, another reporter system named split DNA peroxidase was chosen to replace the FRET system used in the previous design. Clarified sequences of DNA peroxidases and methods for their resolution are described in Shimron, S .; Wang, F .; Orbach, R .; Willner, I. < / RTI > And through the autonomous assembly of the hemine / G-quadruple DNAzyme nanowires (enzyme-free). Amplified DNA detection is described in Anal Chem 2012, 84, 1042-1048.

Satisfactory, if the oligonucleotide is covalently linked, the signal system is also functional for the detection system according to the invention based on hybridization equilibrium.

The long DNA strand of this embodiment includes several regions (Figure 18). Quadruplicate G-quadruplicate, region a plus b representing the original strand A, region b plus region d representing the original strand B, thus containing biotin, and strand S The ring e, the area a * plus the area b * plus the area d * as a flexible hinge derived from the G-quadruplex is three-quarters of the G-quadruplex.

Because of the absence of STV, sat d is longer than the hold soil hold a, d * b * is hybridized first and bd. In this state, if the first bad region b is not effectively forced to bulge, the two G-quadrant portions can not be merged. When there is an STV that binds to biotin on the second region b , the protein lumps interfere with local hybridization or breakpoint movement and pushed into the region b * selected as its preferred partner in the first region b . As a result, the two components of the G- quadruple body (G-quadruplex) is only located in the right position to form a complete G-4 molecules (G-tetrads), with the help of hemin, ABTS ^● in ABTS ^2- H ₂ O ₂ to ^- is found to have the ability for catalyzing the oxidation medium. The kinetic of this reaction can be detected directly by the naked eye or by means of a spectrophotometer, since the product ABTS ^{● -} is green. The structural change process is shown in Fig.

The absorbance of the sample in the presence or absence of STV after 1 hour from the addition of ABTS as a peroxidase substrate is shown in Fig. 18B. An increase of more than 50% after STV addition can be observed. This demonstrates the concept that STV binding causes rearrangement and formation of more G-4 molecules (G-tetrad). Therefore, it was confirmed that biotin was not present in the test sequence and that the signal was not changed in this case.

Review

In this example, the detection system was further simplified by using only one DNA strand labeled with a ligand to perform target protein detection. Moreover, the new catalytic method is used as a reporter as well as an amplification approach. The STV conjugation of this example disturbs the intramolecular equilibrium of the strand displacement exchange reaction and reflects the absorbance of the peroxidase product. We anticipate this system as a potential alternative to an enzyme-linked immunosorbent assay (ELISA) and also target proteins or small molecules (antibody and antigen specific) and use color changes as markers.

Example  13

Effect of toe hold length and position of coupling moiety

Various tests have been performed to optimize the performance of the method (data not provided).

The closeness of the melting temperatures of A and B is known to be important for the sensitivity of the equilibrium. Because A and B share the same split-point moving area, the toh-hold area is a determining factor in the magnitude of the target-coupling effect in this system. The inventors have adjusted the tohield region of A for better identification between STV presence and non-existence. When A has a 2 nt or 3 nt toe, the effect of STV, that is, the relative FRET change, is the largest. The reason why the 4 nt to hold for A is not optimal is that the two strains of B on the system can be changed to equilibrium. Since A has also been modified here, we use a 3 nt to hold for A in the standard setup (Example 1).

Besides thermodynamic, the present inventors have confirmed that the tohole length thus influences the kinetic of the system. The longer to hold specifically increases the reaction rate, which leads to equilibrium much faster. Longer toads cause faster tohold hybridization and are rate-limiting steps in all processes.

The effect of the biotin position is also investigated in this Example. Biotin is labeled at one of three common positions on B phase: the toehold region (TH), the branch migration region (BM), and the 3 'terminal (T3) ). Biotin on the tohole is the most sensitive to STV-binding in terms of FRET signal changes, followed by biotin on BM. Biotin at the 3 'end of B showed only negligible STV effect. DNA branching is valid because it is known to be highly reactive to dysplasia in the presence of magnesium, and therefore small local environmental changes can pose substantial barriers. However, simple hybridization does not have this property.

<110> Gothelf, Kurt Vesterager          Zhang, Zhao <120> Detection of non-nucleic acid analytes using strand displacement          exchange reactions <130> PD10048PC00 <150> DK PA 2012 70555 <151> 2012-09-11 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <400> 1 ctcattcaat accctacg 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <400> 2 ttcaataccc tacgtctc 18 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <400> 3 gagacgtagg gtattgaatg ag 22 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <220> <221> modified_base <222> (9) Int Amino Modifier C6 dT, with Alexa Fluor 647 Succinimidyl Ester <400> 4 tcattcaata ccctacg 17 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <220> <221> modified_base <222> (11) <223> Biotin dT <400> 5 ttcaataccc tacgtctc 18 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <400> 6 tggagacgta gggtattgaa tgaggg 26 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <220> <221> modified_base <10> &Lt; 223 > Amino Modified C6 dT with Alexa Fluor 647 Succinimidyl Ester <400> 7 ctcattcaat accctacg 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <220> <221> modified_base <222> (11) <223> 5-Aminoallyl-dU with digoxigenin <400> 8 ttcaataccc tacgtctc 18 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <220> <221> modified_base <222> (2) <223> 5-Aminoallyl-dU with digoxigenin <220> <221> modified_base <222> (6) <223> 5-Aminoallyl-dU with digoxigenin <220> <221> modified_base <222> (11) <223> 5-Aminoallyl-dU with digoxigenin <220> <221> modified_base &Lt; 222 > (15) <223> 5-Aminoallyl-dU with digoxigenin <400> 9 ttcaataccc tacgtctc 18 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <220> <221> modified_base <10> &Lt; 223 > Amino Modified C6 dT with Alexa Fluor 488 Succinimidyl Ester <400> 10 ctcattcaat accctacg 18 <210> 11 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <220> <221> modified_base <222> (6) <223> Amino Modified C6 dT with Alexa 647 <400> 11 ttcaataccc tacgacctgg gggagtattg cggaggaagg t 41 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <220> <221> modified_base <222> (11) <223> Amino Modified C6 dT with Alexa 555 <400> 12 ccccaggtcg tagggtattg aatgaggg 28 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <400> 13 tgggtcattc aataccctac g 21 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <220> <221> modified_base <222> (11) <223> Biotin dT <220> <221> modified_base <222> (17) <223> Biotin dT <400> 14 ttcaataccc tacgtctc 18 <210> 15 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <400> 15 tggagacgta gggtattgaa tgtgggcggg tgggt 35 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <220> <221> modified_base <222> (11) <223> 5-Aminoallyl-dU with Vitamin D <400> 16 ttcaataccc tacgtctc 18 <210> 17 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence_ <220> <221> modified_base <222> (24) <223> Amino Modified C6 dT with biotin <400> 17 tgggtcaata ccctacgata ccctacgtct cttttttgag acgtagggta ttgtgggcgg 60 gtgggt 66

Claims (19)

1. An analyte detection system for detecting DNA and RNA and other analytes comprising at least a first oligonucleotide A, a second oligonucleotide B, and a third oligonucleotide S,
Each of oligonucleotide A and oligonucleotide B comprises a complementary or partially complementary sequence to the sequence of oligonucleotide S wherein oligonucleotides A and B compete for hybridization with oligonucleotide S in dynamic equilibrium, Optionally, at least one of the oligonucleotides A and B comprises a covalently linked binding moiety capable of interacting with DNA and RNA and other analytes;
The covalently linked binding moiety, which is bound to one or more of the oligonucleotides A and B, or to the oligonucleotide, is capable of interacting with DNA and RNA and other analyte to form the oligonucleotide or binding Wherein the interaction of the moiety and the analyte results in a shift of the hybridization equilibrium and the movement of the equilibrium provides a detectable signal.
2. The analyte detection system of claim 1, wherein the oligonucleotide is a separate nucleotide strand.
2. The analyte detection system of claim 1, wherein the two or more oligonucleotides are partially or fully connected by covalent bonds.
2. The analyte detection system of claim 1, wherein the oligonucleotide S comprises at least one hybridization domain and optionally at least two hybridization domains are located on separate nucleotide strands.
The analyte detection system of claim 1, comprising a first oligonucleotide (1), a second oligonucleotide (3), and a third oligonucleotide (5)
The first or second oligonucleotide comprises a first group (2) forming a first part of the signal system;
The third nucleotide comprises a second group (6) forming a second part of the signal system;
At least one covalently bonded moiety (4) is located on the first or second oligonucleotide;
Wherein the hybridization between the first or second oligonucleotide and the third oligonucleotide can produce a signal or facilitate the signal signal when the first or second oligonucleotide and the third oligonucleotide are not hybridized;
Wherein the presence of the analyte changes the hybridization equilibrium of the detection system to cause a change in signal.
6. The method of claim 5,
The first oligonucleotide (1)
A first tohold region 8 located at the 5'-plane of the branch migration region 7;
The second oligonucleotide (3)
A second toothed region (9) located on the 3'-plane of the minute shift region (7);
The third oligonucleotide (5)
A first tohold region 8 ';
A second toe hold region 9 '; And
&Lt; RTI ID = 0.0 &gt; o &lt; / RTI &gt;
here:
The first to fourth region 8 of the first oligonucleotide 1 and the first toe region 8 ' of the third oligonucleotide 5 comprise a complementary sequence;
The splitting point shifting region 7 of the first oligonucleotide 1 and the splitting point shifting region 7 'of the third oligonucleotide 5 comprise a stretch of complementary nucleotides;
The second toothed region 9 of the second oligonucleotide 3 and the second toothed region 9 'of the third oligonucleotide 5 comprise an extension of complementary nucleotides;
The analyte detection system according to any one of the preceding claims, characterized in that the analyte detection system (1) comprises an extension of complementary nucleotides, wherein the branch point shifting region (7) of the second oligonucleotide (3) and the branch point shifting region (7 ') of the third oligonucleotide .
The method according to claim 1,
- the first oligonucleotide (1) is
A first toothed region 8 located on the 5'-plane of the branch migration region 7;
Optionally one or more covalently bonded moieties (4);
A first group (2) that optionally forms a first part of the signaling system;
- the second oligonucleotide (3) is
A second tohold region 9 located on the 3'-plane of the branching point moving region 7;
Optionally one or more covalently bonded moieties (4);
A first group (2) optionally forming a first part of the signal system;
- the third oligonucleotide (5)
A first tohold region 8 ';
A second tohold region 9 ';
A minute point moving region 7 ';
Optionally one or more covalently bonded moieties (4);
A second group (6) forming a second part of the signaling system;
The first group (2), which conditionally forms the first part of the signal system, comprises a first oligonucleotide (1) and / or a second oligonucleotide (3);
Conditionally, the at least one covalently bonded moiety (4) is located in the first oligonucleotide (1) and / or the second oligonucleotide (3) and / or the third oligonucleotide (5);
Wherein the first to fourth region (8) of the first oligonucleotide (1) and the first to fourth region (8 ') of the third oligonucleotide (5) comprises a complementary sequence;
Wherein the junction point shifting region 7 of the first oligonucleotide 1 and the junction point shifting region 7 'of the third oligonucleotide 5 comprise a stretch of complementary nucleotides;
Wherein the second toe region (9) of the second oligonucleotide (3) and the second toe region (9 ') of the third oligonucleotide (5) comprise an extension of the complementary nucleotide;
Wherein the splitting point shifting region 7 of the second oligonucleotide 3 and the splitting point shifting region 7 'of the third oligonucleotide 5 comprise an extension of the complementary nucleotide;
Wherein the hybridization between the first oligonucleotide (1) and the third oligonucleotide (5) produces a signal or a signal that is different from the case where the first oligonucleotide (1) and the third oligonucleotide (5) Wherein the first group (2), which conditionally forms the first part of the signal system, comprises a first oligonucleotide (1);
Wherein the hybridization between the second oligonucleotide (3) and the third oligonucleotide (5) generates a signal, or the second oligonucleotide (3) and the third oligonucleotide (5) Wherein the first group (2), which conditionally forms a first part of the signal system, comprises a second oligonucleotide (3).
8. A system according to any one of the preceding claims, wherein the signal system formed by the first group (2) and the second group (6) comprises a quencher-fluorophore signaling system, A fiuorophore-quencher signaling system, a FRET signaling system, a DNA peroxidase catalysis signaling system, or a quencher and singlet oxygen sensitizer; And optionally wherein the signaling system uses fluorescent nanoparticles.
9. The analyte detection system according to any one of claims 1 to 8, further comprising hemin and / or ABTS ^{2 -} and / or H ₂ O ₂ and / or luminol.
10. A compound according to any one of claims 1 to 9, wherein the at least one covalently bound moiety (4) is selected from the group consisting of organic molecules, antibodies, antigens, aptamers, biotin and hapten Is selected.
11. The method according to any one of claims 1 to 10, wherein the at least one covalently bonded moiety (4) is selected from the group consisting of 150-1200 Da, 150-1000 Da, 150-800 Da, 150-600 Da, 150-400 Organic molecules having a molecular weight in the range of 150-1500 Da, such as Da, 150-300 Da, 300-1500 Da, 400-1500 Da, 600-1500 Da, 800-1500 Da, 1000-1500 Da, The analyte detection system.
12. The analyte detection system according to any one of claims 1 to 11, wherein the binding moiety has its binding partner bound to the binding moiety.
13. The analyte detection system according to any one of claims 1 to 12, wherein the analyte is selected from the group consisting of proteins, peptides, organic molecules, antibodies, antigens and haptens.
14. The method of any one of claims 1 to 13, wherein the analyte is selected from the group consisting of 150-1200 Da, 150-1000 Da, 150-800 Da, 150-600 Da, 150-400 Da, 150-300 Da, 300- Is an organic molecule having a molecular weight in the range of 150-1500 Da, such as 1500 Da, 400-1500 Da, 600-1500 Da, 800-1500 Da, 1000-1500 Da, or 1200-1500 Da. .
15. The method according to any one of claims 1 to 14, wherein the first oligonucleotide (1) is covalently linked to a second oligonucleotide (3), the second oligonucleotide (3) Wherein the analyte is covalently bound to the nucleotide.
17. A kit of parts comprising an analyte detection system according to any one of claims 1 to 15.
comprising the steps of: a) providing a sample containing or suspected of containing an analyte of interest;
b) providing the analyte detection system of any one of claims 1 to 15;
c) incubating the sample in the analyte detection system;
d) comparing the reference level with the detected level of the analyte; And
e) measuring the presence or level of the analyte in the sample;
&Lt; / RTI &gt; the presence or level of DNA and RNA and other analytes in the sample.
18. The method of claim 17, wherein the hybridization equilibrium between the first oligonucleotide and the third oligonucleotide and between the second oligonucleotide and the third oligonucleotide is dependent on binding of the analyte to the binding moiety and / or release of the binding partner to the binding moiety Lt; / RTI &gt;
19. The method of claim 17 or 18, wherein the sample is a biological sample such as serum or plasma, a urine sample, a feces sample, a biopsy sample, or a saliva sample.

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